Use of nitroflavonoids for the treatment of anxiety

ABSTRACT

Methods of treating anxiety with flavonoid compounds according to Formula (I) and dimers thereof, compounds of Formula (I) and dimers thereof, use of compounds of Formula (I) and pharmaceutical formulations comprising flavonoids of Formula (I) and dimers thereof. ##STR1##

FIELD OF INVENTION

The present invention relates to flavonoids which have been found tohave anxiolytic properties (i.e. anxiety reducing) without correspondingdepression of the central nervous system which is commonly also found inknown sedatives such as benzodiazepines. In particular, the presentinvention relates to flavonoids comprising nitro groups located on thephenyl ring and analogues thereof.

BACKGROUND

Co-pending patent application WO 95/05169 (Strathclyde University)relates to flavonoids and their use in methods of treating anxiety inpatients. The flavonoids of WO 95/05169 are described as havinganxiolytic properties without associated depression of the centralnervous system (e.g. sedative and muscle relaxant effects) commonlyfound with benzodiazepines. The compounds of WO 95/05169 fall under ageneral formula: ##STR2## wherein

R¹, R², R³ and R⁴, R⁵ and R⁸ are independently selected from H, OH, R,NO₂, halo, OR, NH₂, NHR, NR₂, COOR, COOH, CN, or a sugar group;

R⁶ and R⁷ are both H, or R⁶ and R⁷ together form a single bond;

R is C₁₋₆ alkyl or alkenyl; or dimers thereof.

Preferred compounds of WO 95/05169 are described in halo derivatives, inparticular where R⁵ is halo at the 2' position of the above generalformula.

It has now been found that certain compounds falling within the genericformula of WO 95/05169 exhibit unexpectedly good anxiolytic activitywithout associated depression of the central nervous system (e.g.sedative and muscle relaxant effects) commonly found withbenzodiazepines, when the flavone nucleus and/or phenyl ring comprisesat least one NO₂ substituent. Thus, patients may be treated for anxietywithout inducing sedative or myorelaxant side-effects.

It has also been found that compounds of the present invention display asubstantially reduced or no anti-convulsant effect, and that memory isapparently not adversely affected, side-effects commonly found withbenzodiazepines.

STATEMENT OF INVENTION

According to the present invention there is provided a method oftreating anxiety in a patient which comprises administering to thepatient an effective non-toxic amount of a flavonoid of general formula(I): ##STR3## wherein

R¹, R², R³, R⁴ and R⁵ are independently selected from NO₂ and H;

R⁶ and R⁷ are independently selected from Br, Cl, F, and H or R⁶ and R⁷together form a single bond;

R⁸, R¹⁰ and R¹¹ are independently selected from H, --OH, --R, --NO₂, Br,Cl, F, --OR, --NH₂, --NHR, --NR₂, --COOR, --COOH, --CN or a sugar group;

R⁹ is selected from H, NO₂, Br, Cl, or F;

R is C₁ -C₆ alkyl or alkenyl;

with the proviso that at least one of R¹, R², R³, R⁴ and R⁵ is NO₂ andwith the exception that when R⁹ is H, R⁵ is H.

or the administration of an effective non-toxic amount of a bi-flavonoidwhich is a dimer of a compound of general formula (I) and wherein R¹ toR¹¹ and R have the meanings given for general formula (I).

The sugar group may be any of the known sugars, includingmonosaccharides, disaccharides and polysaccharides; and may inparticular be glycosyl, galactopyranosyl or mannopyranosyl.

Preferred compounds of Formula I include compounds wherein

R¹, R² and R⁵ are independently selected from NO₂ and H;

R⁸, R¹⁰ and R¹¹ are all hydrogen;

R⁹ is selected from H, NO₂, Br, Cl and F; with the proviso that at leastone of R¹, R¹ and R⁵ is NO₂ with the exception that when R⁹ is H, R⁵ isH.

More preferred compounds of Formula (I) are those wherein

R¹, R² and R⁵ are independently selected from H and NO₂ ;

R⁶ and R⁷ together form a single bond;

R⁸, R¹⁰ and R¹¹ are all hydrogen;

R⁹ is selected from H, NO₂, Br, Cl and F; with the proviso that at leastone of R¹, R² and R⁵ is NO₂ with the exception that when R⁹ is H, R⁵ isH.

Most preferred compounds of Formula (I) are those wherein

R¹, R² and R⁵ are independently selected from NO₂ and H;

R³, R¹, R⁸, R¹¹ are all H;

R⁶ and R⁷ together form a single bond;

R⁹ is selected from Br, Cl, F and NO₂ ;

with the proviso that at least one of R¹, R² and R⁵ is NO₂.

Examples of preferred compounds of formula (I) for use in treatinganxiety in a patient include:

6,3'-dinitroflavone ##STR4##

6, Bromo, 2'nitroflavone ##STR5##

6, Bromo, 3'nitroflavone ##STR6##

6, Bromo, 4'nitroflavone ##STR7## 3'nitroflavone ##STR8##

4'nitroflavone ##STR9##

6, chloro, 3'nitroflavone ##STR10##

6, fluoro, 3'nitroflavone ##STR11##

Especially preferred are compounds (II) (IV), and (VIII) above.

Compounds wherein R⁶ and R⁷ together form a single bond are flavonederivatives, whereas compounds wherein R⁶ and R⁷ are both H areflavanone derivatives.

The bi-flavonoid is a dimer of two covalently bonded moieties which areeach of general formula (I) as set forth above. Bonding between the twomoieties generally occurs at the 3'-position of one moiety and8-position of the other moiety. The preferred bi-flavonoid has generalformula (X) wherein R¹ to R¹¹ and R have the same meanings as forgeneral formula (I): ##STR12##

The compounds of general formula (X) wherein at least one of R¹ and R²in each of the dimer moieties of general formula (I) is NO₂ ; R⁹ isselected from Cl, Br, F and NO₂ ; R⁶ and R⁷ are both H or R⁶ and R⁷together form a single bond; and all other R functional groups arehydrogen are preferred.

The compounds of general formula (X) wherein the compounds are 3' or4'nitro containing compounds; R⁴ is selected from NO₂, Cl and Br; R⁶ andR⁷ are both H or R⁶ and R⁷ together form a single bond; and all other Rfunctional groups are hydrogen are more preferred.

Pharmaceutical formulations include at least one compound of generalformula (I) and/or (X) together with at least one pharmaceuticallyacceptable carrier or excipient. Naturally, the skilled addressee willappreciate that compounds of the invention employed in pharmaceuticalformulations of the invention possess R groups R¹ to R¹¹ and R asdefined herein. Each carrier must be "pharmaceutically acceptable" inthe sense of being compatible with the other ingredients of theformulations and not injurious to the patient.

It should be understood that the flavonoid compounds of the presentinvention can be administered in the form of pharmaceutically acceptablesalts or esters thereof. Salts are usually acid addition salts (e.g.with hydrohalogen acids) or acceptable metal salts (e.g. Na, Ca, Mg).

Formulations include those adapted for oral, rectal, nasal, vaginal andparenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. Such methods include the step of bringinginto association the active ingredient with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly ad intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then if necessary shaping the product.

Formulations of the present invention adapted for oral administrationmay be presented as discrete units such as capsules, sachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous oron-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxypropylmethylcellulose),lubricant, inert diluent, preservative, disintegrant (e.g. sodium starchglycolate, cross-linked povidone, cross-linked sodiumcarboxymethylcellulose) active-surface or dispersing agent. Mouldedtablets may be made by moulding in a suitable machine a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay optionally be coated or scored and may be formulated so as toprovide controlled release of the active ingredient therein using, forexample, hydroxypropylmethylcellulose in varying proportions to providethe desired release profile.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations for vaginal administration may be presented as pessaries,tampons, creams, gels, pastes, foams or spray formulations containing inaddition to the active ingredient such carriers as are known in the artto be appropriate.

Formulations are parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containanti-oxidants, buffers, bacteriostatis and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose sealed containers, for example, ampoules andvials, and may be stored in a freeze dried (lyophilized) conditionrequiring only the addition of the sterile carrier, for example waterfor injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

The dose will depend on a number of factors known to the skilledphysician including the severity of the conditions, the identity of therecipient; and also the efficacy and toxicity of the particular compoundof general formula (I) which is being administered. Generally doses inthe range 0.1-100 mg/kg body weight may be used, particularly 1-10mg/kg. The frequency of administration will vary depending on the rateof metabolism or excretion of the administered compound, but may berepeated daily, optionally as two or more sub-doses. Unit doses of 20 to500 mg, preferably 100 to 400 mg may be used.

As a further aspect of the present invention there is provided acompound of general Formula (I) ##STR13## wherein

R¹, R², R³, R⁴ and R⁵ are independently selected from NO₂ and H;

R⁶ and R⁷ are independently selected from Br, Cl, F, and H or R⁶ and R⁷together form a single bond;

R⁸, R¹⁰ and R¹¹ are independently selected from H, --R, NO₂, Br, Cl, F,--OR, --NH₂, --NHR, --NR₂, --COOR, --COOH, --CN or a sugar group;

R⁹ is selected from H, NO₂, Br, Cl, or F;

R is C₁ -C₆ alkyl or alkenyl;

with the proviso that at least one of R¹, R², R³, R⁴ and R⁵ is NO₂ andwith the exception that when R⁹ is H, R⁵ is H.

or a bi-flavonoid which is a dimer of a compound of general Formula (I)and wherein R¹ to R¹¹ and R have the meanings given for general Formula(I).

Preferred compounds of Formula (I) include compounds wherein

R¹, R² and R⁵ are independently selected from NO₂ and H;

R⁸, R¹⁰ and R¹¹ are all hydrogen;

R⁹ is selected from H, NO₂, Br, Cl and F; with the proviso that at leastone of R¹, R² and R⁵ is NO₂ and with the exception that when R⁹ is H, R⁵is H.

More preferred compounds of Formula (I) are those wherein

R¹, R² and R⁵ are independently selected from H and NO₂ ;

R⁶ and R⁷ together form a single bond;

R⁹ is selected from H, NO₂, Br, Cl and F; with the proviso that at leastone of R¹, R² and R⁵ is NO₂ and with the exception that when R⁹ is H, R⁵is H.

Most preferred compounds of Formula (I) are those wherein

R¹, R² and R⁵ are independently selected from NO₂ and H;

R³, R⁴, R⁸, R¹¹ are all H;

R⁶ and R⁷ together form a single bond;

R⁹ is selected from Br, Cl, F and NO₂ ;

with the proviso that at least one of R¹, R² and R⁵ is NO₂.

Preferred compounds of the invention include 6, 3'-dinitroflavone and 6halogen, 3'-nitro or 6 halogen, 4'-nitroflavones and derivativesthereof. Examples of preferred compounds of the invention include:

6,3'-dinitroflavone ##STR14## 6, Bromo, 2'-nitroflavone ##STR15##

6, Bromo, 3'nitroflavone ##STR16##

6, Bromo, 4'nitroflavone ##STR17## 3'nitroflavone ##STR18##

4'nitroflavone ##STR19##

6, chloro, 3'nitroflavone ##STR20##

6, fluoro, 3'nitroflavone ##STR21##

Most preferred compounds of the invention are II, IV and VIII.

In a further aspect of the invention there is provided use of compoundsof Formula (I) in the preparation of a medicament for the treatment ofanxiety in a patient. In a preferment there is provided use of acompound according to any one or more of formulae (II), (III) to (IX)and (X) in the preparation of a medicament for the treatment of anxietyin a patient. More preferably there is provided use of a compound or acocktail of compounds selected from compounds (II), (IV) and (VIII) inthe preparation of a medicament for the treatment of anxiety in apatient.

The invention will now be illustrated by reference to the followingfigures.

FIG. 1:

Scatchard plot of representative curves of ³ H-Flunitrazepam (³ H-FNZ)binding to bovine synaptosomal membranes in the absence () or in thepresence of compound II (▪, 20 nM).

FIG. 2:

Performance of mice during a 5 minute test on the elevated plus-mazetest, 15 minutes after i.p. injection with vehicle (VEH) or compound(II) (0.3-100.0 μg/Kg). Results are expressed as mean ±SEM of the numberof total arms entries (hatched bars), percentage of open arms entries(open arms) and percentage of time spent in the open arms (closed bars).*: p<0.05, **: p<0.01, Dunnet's multiple comparison test. The number ofexperimental mice per group used ranged between 9-16.

FIG. 3:

Competition by 6, 3'-dinitroflavone of [³ H]-FNZ binding to extensivelywashed crude synaptosomal membranes from various rat brain regions.Membranes from cerebellum (▪), cerebral cortex (O) and spinal cord (▾)were prepared as described in Material and Methods. Data are from arepresentative experiment replicated 6-8 times.

FIG. 4:

Competition experiments by inhibiting 0.65 nM [³ H]-FNZ binding sagittalsections of the rat brain with various concentrations of6,3'-dinitroflavone. Preparation of brain sections for autoradiographicanalysis was described in Material and Methods. Representativedisplacement curves from cerebellum (▪); parietal cortex (O) and dentategyrus (▾) are shown.

FIG. 5:

Ambulatory locomotor activity counts during a 5-min test session in aOpto-varimex® apparatus 15 min after an i.p. injection of vehicle (VEH)or 6,3'-dinitroflavone (DNF, 0.001-10 mg/kg as detailed). Data areexpressed as mean ±S.E.M. Number of animals in the experimental groupsranged between 10 and 16. *p<0.05, **p<0.01, significantly differentfrom controls (Dunnett's multiple comparison tests after ANOVA).

FIG. 6:

Mean ±S.E.M. of total entries (open bars), percentage of open armentries (hatched bars) and percentage of time spent in open arms (closedbars) of mice given a 5 min session in the elevated plus maze 20 minafter i.p. injection of vehicle (VEH), 6,3'-dinitroflavone (DNF, 30μg/kg) or DNF (30 μg/kg)÷Ro 15 1788 (1 mg/kg). *p<0.01, significantlydifferent from controls (Dunnett't-test after ANOVA). Number of animalsper group is shown in parentheses.

FIG. 7:

Mean ±S.E.M. number of head dips (closed bars) and time (in seconds)spent head-dipping (hatched bars) of mice given a 5 min session in thehole board test 20 min after an i.p. injection of vehicle (VEH) or6,3'-dinitroflavone (DNF, 0.3-10 mg/kg). *p<0.01, significantlydifferent from controls (Dunnett's t-test after ANOVA). Number ofanimals per group is shown in parentheses.

FIG. 8:

Performance of mice in the horizontal wire test after an i.p. injectionof vehicle (VEH) or 6,3'-dinitroflavone (DNF, 0.3-10 mg/kg). The sessiontest took place after two trials, executed after a 5 min interval (seeMaterials and Methods). Number of animals per group is shown inparentheses.

FIG. 9:

Performance of mice in the horizontal wire test 20 min after an i.p.injection of diazepam (DZ, 1 mg/kg) or 6,3'-dinitroflavone (DNF, 1mg/kg)÷diazepam (DZ, 1 mg/kg). The session test took place after twotrials, executed after a 5 min interval (see Material and Methods).*p<0.0001, Chi square frequency test. Number of animals per group isshown in parentheses.

FIG. 10:

Effect of pre-training and post-training i.p. administration of vehicle(open bars) or 100 μg/kg or 6,3'-dinitroflavone (hatched bars) in memoryof an inhibitory avoidance test. The ordinate represents the step-downlatency (in seconds) of the test session. Data are expressed as medians(interquartile range). Number of animals per group is shown inparentheses.

FIG. 11:

Shows that binding of 0.5 nM ³ H-FNZ, to extensively washed crudesynaptosomal membranes from rat cerebellum (), cerebral cortex (O) andspinal cord (▾), was displaced by 9-14 different concentrations ofcompound (IV). Data are from a representative experiment replicated 3-6times. The competition curves were analysed using the Graph-pad software(see results in Table 5).

FIG. 12:

Mean ±S.E.M. of total arms entries (open bars), percentage of open armsentries (closed bars) and percentage of time spent in the open arms(hatched bars) of mice given a 5 min session in the elevated plus-maze,20 min after i.p. injection with vehicle (VEH) or 100 μg/kg of compound(IV). *p<0.01, Student t test. Number of animals per group=17.

There now follow examples which illustrate the invention. It is to beunderstood that the examples are not intended to limit the scope of theinvention in any way.

EXAMPLES SECTION 1 Example 1:

Preparation of 6,3'-Dinitroflavone (II) and 6,4'-Dinitroflavone (IIa)

6,3'-Dinitroflavone (II) and 6,4'-dinitroflavone (IIa) were prepared asfollows (Scheme A): ##STR22##

Anhydrous nitric acid (d=1.4, 750 μL) was added dropwise to flavone (1)(Extrasynthese, France) (60 mg; 0.27 mmol).

The vial containing (1 ) was kept in an ice bath during the addition.The resulting solution was allowed to stand for 30 minutes at roomtemperature. While stirring with a thin glass rod, water (10 mL) wasadded, and the vial placed in an ice bath to cool. The precipitatedproduct was collected by vacuum filtration, washed with water and dried.Its toluene solution was chromatographed in a silica gel column whichwas eluted in steps with increasing concentrations of acetone intoluene. Two major components could be isolated which were furtherpurified by recrystallisation from acetone-water rendering compounds(II) and (IIa). Compound (II): yield 45%; yellow light crystals (fromacetone-water);

mp 246-248° C.; ¹ H NMR (300 MHz, DMSO-d₆): δ 8.90 (t, J=2 Hz, 1H), 8.72(d, J=2.6 Hz, 1H), 8.64 (dd, J=9.3 and 2.6 Hz, 1H), 8.59 (m, 1H), 8.46(m,1H), 8.16 (d, J=9.3 Hz, 1H), 7.89 (t, J=8.4 Hz, 1H), 7.43 (s, 1H).EIMS m/z 312 [M]⁺, 284, 266, 238, 220, 165.

Compounds (IIa) yield 45%, yellow crystals (from acetone-water);

mp 260-261° C.; ¹ H NMR (300 MHz, DMSO-d₆); δ 8.72 (s, 1H), 8.65 (d,J=9.1 Hz, 1H), 8.40 (m, 4H), 8.10 (d, J=9.2 Hz 1H), 7.42 (s, 1H). EIMSm/z 312 [M]⁺, 284, 266, 254.

RESULTS

Non-specific nitration of the flavone nucleus yielded the two nitratedflavone (II) and (IIa). Compounds (II) and (IIa) inhibited ³H-Flunitrazepam (³ H-FNZ) binding extensively washed bovine cerebralcortical membranes with a Ki of 12.0±1.7 nM (n=7) and 17±5 μM (n=3),respectively. Briefly, ³ H-FNZ binding was carried out as described byLevy de Stein, M., Medina, J. H., De Robertis E., Mol. Brain Res. 1985,5, 9-15. In brief, for each assay, triplicate samples of the membranes,containing 0.2 to 0.4 mg protein were suspended in a final volume of 1mL of 0.25 mM Tris-HCl buffer, pH 7.3. The incubation was carried out at4° C. for 60 minutes with 0.6 nM ³ -FNZ. To study the bindingsaturation, a range of 0.3 to 10 nM ³ H-FNZ was used. Non-specificbinding was determined in parallel incubations in the presence of 3 μMFNZ, and represented 5-15% of total. The assays were terminated byfiltration under vacuum though Whatman GF/A glass-fiber filters, andthree washes with 3 mL each of incubation medium. Filters were dried andcounted after the addition of 5 mL of 2,5-diphenyloxazole/xylene asscintillation fluid. Bovine cerebral cortical membranes were treatedsubstantially following the method of Medina, J. H., De Robertis, E. J.,J. Neurochem. 1985, 44, 1340-1345. Scatchard analysis of saturationcurves for compound (II) revealed a competitive interaction showing adecline in the apparent affinity without changes in the maximal numberof sites (Bmax) (FIG. 1). Compound (II) showed a very high affinity forthe benzodiazepine (BDZ-R) receptor.

Example 2

Pharmacological Activity: Performance of Mice on the Elevated Pluz-Maze.

As compound (II) showed a very high affinity for the BDZ-R it wasfurther examined for pharmacological activity. Performance of mice onthe elevated plus-maze test was used to measure anxiolytic actions inrodents, following i.p. administration of vehicle or compound (II).

The animals used in the pharmacological test were male Swiss mice frombreeding stock, weighing 28-35 g. They were placed n groups of ten withfree access to water and food, and maintained on 12 h/12 h day/nightcycle. In all the tests the mice were i.p. injected with VEH or asolution of the drug, 15 minutes before the assay.

The elevated plus-maze set-up consisted of a maze of two open arms, 25×5cm, crossed by two closed arms of the same dimensions, with free accessto all arms from the crossing point. The closed arms had walls 35 cmhigh all around. The maze was suspended 50 cm from the room floor. Micewere placed on the central part of the cross facing an open arm. Thenumber of entries and the time spent going into open and closed armswere counted during 5 minutes. A selective increase in the parameterscorresponding to open arms reveals an anxiolytic effect. The totalexploratory activity (number of entries in both arms) was alsodetermined. Results are shown in FIG. 2.

Compound (II) at doses ranging from 0 to 30 μg/kg increased thepercentage of entries in the open arms, without affecting the total armentries. It is important to stress that for diazepam a minimum dose of30 μg/kg is necessary in order to produce similar anxiolytic effects(data not shown). At a dose of 3 and 30 μg/kg, (II) also enhanced thepercentage of the time spent in the open arms (FIG. 2).

In conclusion, it appears that (II) is a very potent anxiolytic drugthat interacts competitively and with high affinity with the BDZ-R.

Comparative anxiolytic concentration ranges of halo derivatives of WO95/05169 and diazepam to compound (II) are shown below:

                  TABLE 1                                                         ______________________________________                                        Compound     Anxiolytic Concentration (μg/kg)                              ______________________________________                                        Compound (II)                                                                              0.3-30                                                           6 Bromoflavone                                                                              500-3000                                                        6,8 dibromochrysin                                                                         1000-3000                                                        2'chlorochrysin                                                                            1000                                                             2'fluorochrysin                                                                            1000                                                             Diazepam      30-1000                                                         ______________________________________                                    

EXAMPLES SECTION 2

Materials and Methods

Animals

Adult male Wistar rats weighing 250 g were used for biochemicalexperiments. Adult male Swiss male weighing 25-30 g were used forpharmacological assays except for inhibitory avoidance and tail flicktests were done in rats. Animals were housed in a controlledenvironment, with free access to food and water and maintained on a 12h/12 h day/night cycle.

Radioreceptor Binding Assays

Displacement curves were performed using [³ ]-FNZ or [³ H]-zolpidem ([³H]-ZOLP) as radioligands in washed crude synaptosomal membranes from ratcerebral cortex, cerebellum, hippocampus, striatum or spinal cord.Membrane preparations were carried out according to Medina et al.(1990). Briefly, brains were rapidly dissected out on ice and thedifferent structures were homogenized in 10 volumes of 0.32 M sucroseand centrifuged at 900×g for 10 min. The resulting supernatant wascentrifuged at 100,000×g for 30 min and the pellet washed twice in 25 mMTris HCl buffer pH 7.4 at 100,000×g for 30 min, and stored at -20° C.until used.

For [³ H]-FNZ (84 Ci/mmol, NEN) displacement curves, differentconcentrations of DNF (0.3 nM to 1 μM) were added to 0.3 mg membraneprotein suspended in 1 ml of 25 mM Tris HCl buffer in presence of 0.6 nMof the radioligand. Protein determination was carried out by using themethod of Lowry et al. (1951). Nonspecific binding (<5%) was determinedin parallel incubations with 10 μM FNZ (Hoffmann-La Roche). Theincubation was carried out at 4° C. for 2 h. The assays were determinedby filtration under vacuum through Whatman GF/A glass fiber filters, andtwo washes with 3 ml each of incubation medium. Filters were dried andcounted after the addition of 5 ml 2,5-diphenyl-oxazole (PPO)-xylene asscintillation fluid.

For [³ H]-ZOLP (50.8 Ci/mmol, NEN) binding assays we used the techniqueof Arbilla et al. (1986), slightly modified as follows: displacementcurves were done with 1 nM of the radioligand and 10 μM zolpidem todetermine the nonspecific binding (<15%). The incubation of the samples(0.4 mg protein in 1 ml of 50 mM Tris HCl buffer, pH 7.4, 120 mM NaCl, 5mM KCl) was carried out at 4' C. during 30 min. The reaction was stoppedwith 3 ml of the same buffer and 3 washes. The other steps were similarto the original technique.

Additional binding studies were performed as described elsewhere: [³]-prazosin binding for α₁ adrenergic receptors (Medina et al., 1984), [³H]-dihidroalprenolol binding for β adrenergic receptors (Medina et al.1984), [³ H]-quinuclinidyl benzylate binding for muscarinic cholinergicreceptors (Jerusalinksy et al., 1983), [³ H]-muscimol binding forGABA.sub.λ receptors (Medina et al., 1983) and [³H]-8-Hydroxydipropylaminotetralin ([³ H]--OH--DPAT) binding forserotonin (5-HT_(l)λ) receptors (Nenonene et al., 1984).

Autoradiographic Experiments

Wistar rats were decapitated and brains rapidly removed. Sagittalsections (15 μm in thickness) were prepared at -20° C. using amicrotome-cryostat. The tissue slices were kept frozen at -70° C. untilused. For DNF displacement curves to [³ H]--FNZ binding (0.65 nM),tissue sections were incubated for 60 min at 4° C. in 25 mM Tris HClbuffer, pH 7.4, in presence of different concentrations of DNF (1-600nM). For non-specific binding we used 10 μM FNZ. The incubation wasterminated by rinsing the sections for two min in cold buffer. Sectionswere briefly dipped in cold distilled water and dried rapidly under astream of cold air (Niddam et al., 1987).

Autoradiograms were generated by apposing the slide-mounted tissuesection to tritium-sensitive film (Hyperfilm Amersham) in a light-proofX-ray cassette at 4° C. for two weeks.

The optical densities from different brain regions were converted firstto radioactive units and then to fmol/mm² using the [³ H] standards onthe film with the aid of a computerized image densitometric analysissystem (MCID 4.02). The values were normalized and the K_(i) values weredetermined using a computerized program (Graph-Pad Prism) Bernabeu etal., 1995; Cammarota et al., 1995).

Pharmacological Procedures

Locomotor Activity Test

An Opto-varimex® apparatus was used according to Viola et al. (1994).The apparatus discriminates between total and ambulatory activities. Anincrease in the number of transitions through the beams reflectsaugmented locomotor activity. In this and all following tests in mice,animals were i.p. injected with the vehicle, or with DNF 20 min beforethe beginning of the tests. In each session, control mice were tested inparallel with those animals receiving drug treatment.

Elevated Plus Maze Test

The test was performed in the same session immediately after thelocomotor activity measurement (Viola et al., 1994; Wolfman et al.,1994). This test is widely validated for rodents (Pellow et al., 1985,Pellow and File, 1986; Lister, 1987) and possesses several advantagesover other tests for measuring anxiety (Dawson and Tricklebank, 1995). Aselective increase in the number of entries in the open arms and thetime spent in the open arms reveals an anxiolytic effect of the drug(Pellow et al., 1985; Pellow and File, 1986). A series of experimentswith the injection of the selective central benzodiazepine receptorantagonist Ro 15-1788 (File and Pellow, 1986) was also carried out.

Holeboard Test

The test was performed according to Viola et al. (1994) and Wolfman etal. (1994). The number of head dips and the time spent head dipping werecounted during 5 min. A decrease in these parameters reveals a sedativebehaviour (File and Pellow, 1985).

Horizontal Wire Test

This test was carried out as previously described (Viola et al., 1994;Wolfman et al., 1994; Viola et al., 1995). The test took place after twotrials, performed at 5 min intervals. A myorelaxant drug will impairmice to grasp the wire (Bonetti et al., 1982). The effect of DNF )1mg/kg) on DZ-induced myorelaxation was also determined.

Sodium Thiopental--Induced Sleeping Time

Sodium thiopental (Abbott) (22 mg/kg) was i.p. injected 15 min aftervehicle or DNF. The disappearance and reappearance of the rightingreflex were considered indications of latency and duration of sleep,respectively (Anca et al., 1993).

Seizure Testing

The effects of DNF on pentylenetetrazole (PTZ)--induced convulsions wereevaluated according to Medina et al., (1990) with slight modifications.PTZ (200 mg/kg) was administered i.p. to mice 15 min after injection ofdrug or vehicle. The number of mice presenting clonic convulsions wasdetermined.

Inhibitory Avoidance Test

This test was performed according to Izquierdo et al., (1990). Thetraining apparatus was a 50×25×25 cm acrylic box with a frontal glasspanel and a floor made of parallel 1 mm caliber bronze bars spaced 0.8mm apart. A 5 cm high, 7 cm wide formica platform was placed on the loftextreme of the box. Rats were placed on the platform and their latencyto step-down placing their four paws on the grid was measured. Onstepping-down they received a 0.35 mA, 2 s scrambled footshock and werewithdrawn from the box (training session). The test session was carriedout 20 h later and was similar to the training session in all respectsexcept that the footshock was omitted. Test step-down latency (to aceiling of 180 s) was taken as a measure of retention of inhibitoryavoidance (Izquierdo et al., 1990).

Tail-Flick Test

This test was performed according to Siegfried et al. (1987). Analgesiawas assessed with a tail-flick apparatus. Rats were wrapped in a toweland placed on the apparatus; the light source positioned below the tailwas focused on a point 2.3 cm rostral to the tip of the tail. Deflectionof the tail activated a photocell and automatically terminated thetrial. Light intensity was adjusted so as to obtain a baselinetail-flick latency (TFL) of 3-6 s. A cut-off time of 10 s was used toprevent tissue damage. Briefly, the general procedure was as follows: abaseline TFL value was obtained for each animal. Following this, therats were placed alone in a waiting cage. TFL value was measured 1 hafter an i.p. injection of vehicle or DNF.

Drugs

6,3'-Dinitroflavone (DNF) and DZ (Hoffman-La Roche) were dissolved indimethylsulfoxide 20%, ethanol 20%, in distilled water, Ro 15-1788(Hoffman-La Roche) was dissolved in propyleneglycol 10% anddimethylsulfoxide 15% in distilled water. The volume of injection was0.1 ml/10 g in mice and 0.1 ml/100 g in rats.

Statistical Analyses

The competition curves were analysed using the Graph-Pad Prism software.Analysis of variance (ANOVA) was used when several treatments in micewere compared. Post-hoc comparisons between individual treatment andcontrols were made using Dunnett's multiple comparisons test. Chi squarefrequency test was used when required. Non-parametric Mann-Whitney Utest was used for inhibitory avoidance and TFL tests in rats.

RESULTS

Biochemical Studies

DNF had different potencies in displacing [³ H]--FNZ binding in variousCNS regions (Table 2). It was more potent in the cerebellum, with aK_(i) value of 17 nM; least potent in the striatum and spinal cord, withK_(i) values of about 44-48 nM; and intermediate in potency in thecerebral cortex and hippocampus. FIG. 3 shows representativedisplacement curves of [³ H]--FNZ binding to cerebellar, cerebralcortical and spinal cord membranes by DNF (9-14 differentconcentrations). In contrast, the non-selective central benzodiazepinereceptor agonist, DZ, displaced with similar K_(i) the [³ H]--FNZbinding to all the brain regions studied (˜7 nM).

Using [³ H]--ZOLP as the radioligand, a well known centralbenzodiazepine receptor agonist that recognizes preferentially the typeI (Arbilla et al., 1986), DNF showed similar K_(i) values insynaptosomal membranes from cerebral cortex, striatum and cerebellum(17.2±3.7 nM, n=5;21±nM, n=2 and 17±1.5 nM, n=3, respectively).

DNF (20 μM) did not displace the binding of [³ H]-quinuclinidylbenzilate, [³ H]-muscimol, [³ H]-prazosin, [³ H]-dihidroalprenolol and[³ H]-8-HO--DPAT to cholinergic muscarinic, GABA.sub.λ α_(l) and βadrenergic and 5-HT_(l)λ receptors, respectively (data not shown).

Similar region variations in the potency of DNF in displacing [³ H]--FNZbinding were observed in autoradiographic experiments (FIG. 2). Themaximal inhibitory effect was observed in the cerebellum (K_(i) 17.2±2.3nM, n=3) followed by parietal cortex (K_(i) 30.1±2.6 nM, n=3), striatum(K_(i) 53.7±7.3 nM, n=3) and dentate gyrus (K_(i) 82.2±7.6 nM, n=3).

Therefore, DNF is 5 times more potent to displace [³ H]--FNZ bindingfrom the cerebellum than from the dentate gyrus.

                  TABLE 2                                                         ______________________________________                                        Inhibition constants of 6,3'-dinitroflavone displacing                        [.sup.3 H-FNZ binding to crude synaptosomal membranes from                    different regions of the rat CNS.                                                                   [.sup.3 H]-FNZ binding                                  Brain regions   n.sup.a                                                                             K.sub.i ± S.E.M., nM                                 ______________________________________                                        Cerebellum      6     17.2 ± 1.9                                           Cerebral Cortex 8     25.9 ± 3.1                                           Hippocampus     4     36.1 ± 5.0                                           Striatum        7     44.1 ± 4.9                                           Spinal Cord     4     48.1 ± 5.5                                           ______________________________________                                         K.sub.i values were obtained by GraphPad Prism software from displacement     curves of [.sup.3 HFNZ binding using 9-15 concentrations of                   6,3dinitroflavone.                                                            .sup.a number of independent experiments.                                

Pharmacological Experiments

Effect of DNF on Ambulatory Locomotor Activity

FIG. 5 shows that the i.p. administration of DNF (up to 3 mg/kg) had noeffect on spontaneous ambulatory locomotion; at 10 mg/kg (the highestdose tested), there was a 55% reduction in the locomotion (F.sub.(9.152)=4.52, p<0.01. A slight increase in this parameter was observed at 0.3mg/kg (p<0.05.

Effect of DNF in the Elevated Plus Maze Test

Previous experiments from our laboratory demonstrated that i.p.injection of low doses (1-30 μg/kg) of DNF in mice had anxiolytic effectas measured in this test. Confirming these results, i.p. administrationof an anxiolytic dose of DNF (30 μg/kg) increased the percentage ofentries in the open arms (F.sub.(2.22) =13.37, p<0.01; Dunnett testafter ANOVA) (FIG. 6). No differences were observed in the total armentries (F.sub.(2.22) =0.83, p<0.05). This anxiolytic effect was blockedby the injection of Ro 15-1788, a specific central benzodiazepinereceptor antagonist (FIG. 6). Experiments run in parallel using DZ,revealed that this well known anxiolytic drug produced an increase inthe percentage of open arm entries only at doses 10-100 times higher(vehicle=22.9±2.0%; DZ 0.3 mg/kg=35.0±4.5%, p<0.05; FIG. 6).

Effect of DNF in the Holeboard Test

Performance of mice injected with vehicle or DNF in the holeboard testis shown in FIG. 7. As can be seen, doses up to 3 mg/kg did not changethe number of head dips and the time spent head-dipping. Only at thehigh dose of 10 mg/kg (300 times higher than the anxiolytic dose used inthis study), DNF decreased both parameters (F(head dips).sub.(2.01)=6.04, F(time).sub.(2.01) =4.63, p<0.01; Dunnett comparison test afterANOVA). DZ provoked similar effects when injected at 1 mg/kg (head dips,vehicle=8.5±1.2; DZ 1 mg/kg=1.2±0.5, p<0.001).

Effect on DNF in the Horizontal Wire Test

DNF, at doses up to 10 mg/kg, did not affect the percentage of micegrasping the wore (FIG. 8). On the other hand, the full centralbenzodiazepine receptor agonist DZ (1 mg/kg) produced a markedmyorelaxant effect (8 animals out of 10) (FIG. 9). This myorelaxantaction was counteracted by the administration of DNF plus DZ (1 mg/kg; 2animals out of 15, p<0.001, X² test) (FIG. 9).

Effect of DNF on PTZ--Induced Convulsions

DNF, in a wide range of doses (30 μg/kg-6 mg/kg), did not prevent theseizures induced by 200 mg/kg PTZ in the mice (Table 3). In contrast, DZ(0.3-3 mg/kg) showed anticonvulsant activity (p<0.001, X² test) (Table3).

                  TABLE 3                                                         ______________________________________                                        Effect of diazepam and 6,3'-dinitroflavone on                                 pentylenetetrazole - induced seizures.                                                             % of convulsing                                                        n.sup.a                                                                              mice                                                     ______________________________________                                        VEH + PTZ       25        96                                                  DZ 0.3 mg/kg     6        33.3.sup.b                                          DZ 1.0 mg/kg    12        42.sup.b                                            DZ 3.0 mg/kg     9        0.sup.b                                             DNF 0.03 mg/kg   6       100                                                  DNF 0.1 mg/kg   12        75                                                  DNF 0.3 mg/kg   15        80                                                  DNF 1.0 mg/kg   13        92.4                                                DNF 3.0 mg/kg   5        100                                                  DNF 6.0 mg/kg   7        100                                                  ______________________________________                                    

Mice were i.p. injected with 200 mg/kg of pentylenetetrazole (PTZ) 15min after an i.p. administration of vehicle (VEH), diazepam (DZ) or6,3'-dinitroflavone (DNF). Data are expressed as the percentage of micepresenting clonic or tonic-clonic seizures. ³ number of animals pergroup.

    .sup.b p<0.001, X.sup.2 test.

Effect on DNF on Thiopental--Induced Sleeping Time

As can be observed in Table 4, i.p. administration of 3 mg/kg DNFaugmented the sleeping time (p<0.05). No changes were observed inlatency to sleep. When injected at a lower dose (1 mg/kg) DNF did notchange the latency or the sleeping time.

                  TABLE 4                                                         ______________________________________                                        Effect of 6,3'-dinitroflavone on thiopental-induced                           sleeping time.                                                                         n.sup.a                                                                              Latency(s)  Sleeping Time(s)                                  ______________________________________                                        VEH + TP   16       233 (195/325)                                                                              190 (11/478)                                 DNF 1 mg/kg + TP                                                                          7       230 (164/300)                                                                              207 (20/600)                                 DNF 3 mg/kg + TP                                                                         12       237 (202/293)                                                                             1277 (248/1800)*                              ______________________________________                                    

Mice were injected with sodium thiopental (TP, 22 mg/kg i.p.) 15 minafter vehicle (VEH) or 6,3'-dinitroflavone (DNF). The ceiling ofsleeping time was 1800 s. Data are expressed in medians (interquartilerange). ³ number of animals per group.

*p<0.05, Dunn's multiple comparison test, after Kruskall Wallis(KW=6.93).

Effect of DNF on Inhibitory Avoidance and Tail Flick Tests

In rats, i.p. administration of 100 μg/kg DNF had no effect either preor posttraining on the test session performance of inhibitory avoidance(FIG. 10). Furthermore, DNF at the same dose, did not alter TFL(Vehicle=3.2 s (2.8/4.3, n=11); DNF 100 μg/kg=3.5 s (2.7/4.2, n=11)[median (interquartile, p<0.05, Mann-Whitney U Test].

DISCUSSION

DNF has anxioselective properties and is thought to act at the centralbenzodiazepine receptor as a partial agonist with low selectivity forthe central benzodiazepine receptor subtypes I and II.

Pharmacological and biochemical evidence suggests the existence of thesetwo distinct central benzodiazepine receptor types (Seigharth andKarobath, 1980; Trifiletti et al., 1984; Niddam et al., 1987; Mohler etal., 1995; McKernan and Whiting, 1996). The type I is the most abundantin the brain. The cerebellum is primarily enriched in this type (Niddamet al., 1987); the hippocampus (Arbilla et al., 1986) and cerebralcortex contain mixed amounts of types I and II (Trifiletti et al, 1984), whereas type II is predominant in a few brain areas such as striatum,spinal cord, dentate gyrus and olfactory bulb (Watanabe et al., 1985;Niddam et al., 1987; Pritchett et al., 1989; McKernan and Whiting,1986). Using crude synaptosomal membranes (FIG. 3 and Table 2), we foundthe lowest DNF inhibition constants for [³ H]--FNZ binding incerebellum, followed by cerebral cortex>hippocampus>striatum˜spinalcord. In autoradiographic experiments the rank order of potency wascerebellum>parietal cortex>striatum>dentate gyrus (FIG. 4). Thesefindings could be due to different affinities of DNF for the centralbenzodiazepine receptor subtypes and their relative densities in eachbrain region. In those areas with mixed populations of subtypes I and II(e.g. cerebral cortex and hippocampus) the resultant K_(i) values mayreflect the average affinity for central benzodiazepine receptor types Iand II. In contrast, we found two well-defined binding sites in corticalmembranes using the preferentially central benzodiazepine receptor typeI agonist CL 218,872 (high affinity K_(i) value=10 nM low affinity K_(i)value=1.2 μM). In addition, using a selective central benzodiazepinereceptor I ligand, [³ H]--ZOLP (Arbilla et al., 1986), DNF had similarK_(i) values for cerebral cortex, striatum and cerebellum. Theseaffinities are concordant with the nature of the cerebellar bindingsite. DNF is a selective ligand for the central benzodiazepine receptorsbecause it did not displace the binding of specific [³ H] radioligandsto α1 and β adrenergic, muscarinic cholinergic, GABA.sub.λ or 5-HT_(l)λreceptors.

Confirming and expanding recent findings indicating that very low dosesof DNF (1-30 μg/kg) have a potent anxiolytic action as measured in theelevated plus maze, the DNF-induced increase in open arms explorationobserved in the present study was blocked by the administration of theselective benzodiazepine antagonist Ro 15-1788 (FIG. 6). DNF (up to 10mg/kg) did not evidence anticonvulsant, myorelaxant, amnesic oranalgesic effects (Table 3 and FIGS. 8 and 10).

On the other hand, DNF possesses a slight depressant action at highdoses (100-300 times higher than those producing anxiolytic effects) asevidenced by the thiopental sleeping time potentiation (Table 4) and byboth a reduction in the locomotor ambulatory activity and a decrease inholeboard exploration (FIGS. 5 and 7) (File 1985). Therefore, DNF canreduce anxiety at doses well below those causing sedation.

In comparison to DZ, DNF is a 30 times more potent anxiolytic andrequired a 10 fold higher dose to produce similar sedative effects.

We demonstrated that DNF was able to reverse the myorelaxant effect ofthe full agonist DZ (FIG. 9).

Due to its selective pharmacological profile and low intrinsic efficacy,and its potentiality to induce less unwanted side effects, DNF mayrepresent an improved therapeutic tool for the treatment of anxiety.

In conclusion, DNF is a specific and high affinity benzodiazepinereceptor ligand that exhibits mild regional differences in its potencyto displace [³ H]--FNZ binding to central benzodiazepine receptors. Ithas an anxioselective action in mice and prevents the muscle relaxationeffect of a full benzodiazepine receptor agonist.

EXAMPLES SECTION 3: 6-BROMO-3'-NITROFLAVONE AND FURTHER NITROFLAVONES

6-bromo-3'-nitroflavone (IV), 6-bromo-2'-nitroflavone (III) and6-bromo-4'-nitroflavone (V) were prepared as follows:

Method A

Compounds IV, V and III were prepared in two steps staring withflavanone (1) (Extrasynthese, France) as indicated in Scheme 1.

To a solution of (1) (2.7 mmol) and pyridine (0.15 mmol) in C Cl₄ (9.6mL), at 0° C., a solution of bromine (6.2 mmol) in C Cl₄ (3.72 mL) wasadded dropwise. The mixture was stirred for 1 h at 30° C. followed by 45min at 65° C. After cooling, the reaction mixture was washed with two100 mL portions of a saturated aqueous solution of Na₂ S₃ O₅ and thenwith water, dried over Na₂ SO₂ and concentrated to dryness in a rotaryevaporator.

The crude product dissolved in toluene was chromatographed in a column(2.5 cm×40 cm) of silica gel, 10-40 μ, type H, eluted stepwise with 200mL of toluene, followed by equal volumes of toluene containing 1, 2, 3,4 and 5% acetone (V/V).

The fractions obtained were pooled according to the results of theiranalysis by TLC. The pool containing 6 bromoflavone (2) was recovered byevaporation of the solvent and recrystallised from ethanol-water.

Compound (2):6 bromoflavone

mp:189-190° C. UV λ_(max) 256, 299 nm. EIMS M⁺ 300 and 302 (C₁₅ H₀ O₂Br). ¹ H NMR (DMSO-d₀, 400 MHz) δ 8.11-8.13 (3 H, m, H-5, H-2', H-6'),8.00 (1 H, dd, J=8, 2 Hz, H-7), 7.47 (1 H, d, J=8 Hz, H-8), 7.57-7.63 (3H, m, H-3', H-4', H-5'), 7.11 (1 H, s, H-3). ^(--C) NMR (100 MHz) 162.9(s, C-2), 106.9 (d, C-3), 175.9 (s, C-4), 124.9 (s, C-4a), 126.9 (d,C-5), 117.9 (s, C-6), 136.9 (d, C-7), 121.3 (d, C-8), 154.7 (s, C-8a),(s, C-1'), 126.5 (C-2'/C-6'), 129.1 (C-3'/C-5'), 132.0 (C-4').

Compound (2) was dissolved in anhydrous nitric acid at 0° C. Theresulting solution was allowed to stand 45 min at room temperature andthen an excess of water was added, with stirring. The precipitatecollected by filtration, was dissolved in toluene and chromatographed,as indicated before, in a column of silica gel: three fractions wereobtain. They were recovered by evaporation of the solvent, purified byrecrystallisation from acetone-water. The compounds obtained wereidentified by ¹ H NMR as those numbered IV, V and III in Scheme 1.##STR23##

The three compounds gave MS and elemental analytical data consistentwith their structures. Compound (IV): ¹ H NMR (300 Mhz, CDCl₃): δ 8.80(t, J=2.0 Hz, H-2'), 8.42 (dt, J=8.0, 2.4 Hz, H-4'), 8.37 (d, J=2.4 Hz,H-5'), 8.21 (dt, J=8.0, 2.0 Hz, H-6'), 7.84 (dd, J=8.8, 2.5 Hz, H-7'),7.76 (t, J=8.2 Hz, H-5'), 7.54 (d, J=9.2 Hz, H-8'), 6.92 (s, H-3').

Compound (V); ¹ H NMR (300 Mhz, CDCl₃){ δ 8.37-8.42 (m, H-5, H-2',H-6'), 8.10 (d, J=8.8 Hz, H-3', H-5'), 7.84 (dd, J=9.0, 2.4 Hz, H-7),7.52 (d, J=9.0 Hz, H-8), 6.92 (s, H-3).

Compound (III): ¹ H NMR (300 Mhz, CDCl₃): δ 8.37 (d, J=2.4 Hz, H-5),8.11 (dd, J=1.5, 7.7 Hz, H-6'), 7.75 (m, H-7, H-3', H-4', H-5'), 7.29(d, J=9.0 Hz, H-8), 6.60 (s, H-3).

The 6,3' dinitroflavones of the invention may also be synthesisedaccording to the teaching of Ares J. J. et al. J. Med. Chem (1995) 38,pp. 4937-4943 (method B) modified as outlined below: ##STR24##

For the purposes of the present invention, the ring numbering system ofScheme II is used for reasons of consistency with respect to thenumbering system of Formula (I).

Naturally, the man skilled in the art will appreciate that othernitroflavones of the invention may be synthesised following the outlineabove when the benzoyl halide (B) comprises one or more nitro groupslocated at other positions on the aromatic ring to the 3' position shownabove, such as at positions 2', 4', 5' and/or 6' of the benzoyl halide.Suitable benzoyl halides include benzoyl chloride, benzoyl bromide orbenzoyl fluoride having the nitro group located at the 3' position orother benzoyl halides wherein the nitro group is positioned at otherpositions on the aromatic ring, for example, at the 2' and/or 4'positions thereon.

The man skilled in the art will also appreciate that halo nitroflavonesof the invention may be synthesised following the general outlineprovided above prior to the nitration step of the flavone nucleus,starting with a suitably halogenated 2-hydroxyacetophenone which maythen be reacted with a suitable benzoyl halide as described above. Halogroups may be positioned on the acetophenone at one or more of the freecarbon positions of the aromatic ring, such as at the 5, 6, 7, and/or 8positions shown above. The halo substituent on the aromatic ring of theacetophenone may be selected from Cl, Br, or F and is preferably locatedat the 6 position of the aromatic ring of the said acetophenone.

In cases wherein nitroflavones of the invention (e.g. compounds VI andVII) do not possess halo or nitro groups on the flavone nucleus, thegeneral synthetic process for producing such nitroflavones may comprisereacting a 2-hydroxyacetophenone comprising H substituents on thearomatic ring at positions 5, 6, 7 and 8 thereof with an appropriatebenzoyl halide and following the general outline above to compound (C).Naturally, the man skilled in the art will appreciate that nitro groupsmay be located on the 3', 4', 5' or 6' position of the phenyl ring ofsuch nitroflavones depending on the design.

To obtain dinitroflavone derivatives with a NO₂ group at position 6,compound (C) comprising H substituents at all free carbon positions ofthe flavone nucleus and appropriate NO₂ substituent on the phenyl ringthereof may be nitrated according to the teaching of Cushman M. et al.J. Med Chem. (1994) 37, pp. 3353-3362.

Specific compounds of the invention i.e. compounds (II) to (IX)inclusive may be prepared as follows:

(1) Compounds 6,3'-dinitroflavone (II) and 3' nitroflavone (VI)

Reference is made to Scheme II.

Step 1

1 To a solution of the acid chloride (B) (Aldrich) (15 mmol) in pyridine(10 ml), at 0° C., solid 2-hydroxyacetophenone (2) (9 mmol) was addedwith stirring. The reaction mixture was stirred for 15 minutes at 0° C.followed by 30 minutes at room temperature. Then it was poured into a 3%aqueous HCl/ice solution with vigorous stirring. The resultingprecipitate was filtered and washed with water. The crude material wasrecrystallized from methanol yielding compound (3) (75% yield).

Step 2

To a solution of (3) (10 mmol) in pyridine (10 ml), at 50° C.,pulverized potassium hydroxide (15 mmol) was added. The mixture wasstirred for 15 minutes and, after cooling, at 10% aqueous acetic acidsolution was added. The resulting precipitate was filtered. The crudematerial, containing the diketone (4), was used in the next step withoutpurification.

Step 3

A mixture of the crude material from step 2 (equivalent to 10 mmol ofcompound (4), with concentrated sulfuric acid (0.5 ml), and glacialacetic acid (13 ml) was heated at reflux for 1 hour and cooled to roomtemperature. Then, the mixture was poured onto crushed ice (75 g), andthe resulting precipitate was filtered.

Recrystallisation from acetone afforded product (C), 3'-nitroflavone(VI) (mp. 205.2-205.7° C.) (65% yield).

Step 4

to a mixture of (C) (5 mmol) in concentrated sulfuric acid (14 ml), atroom temperature, nitric acid (d=1.40, 1.6 ml) was added with stirring.The reaction was allowed to proceed for 3 hours, and the mixture wasthen poured onto ice (100 g). The precipitated product was filtered,washed with water, and dried. Recrystallisation from acetone afforded(5), 6,3'-dinitroflavone (II) (mp. 290-292° C., yield 90%; NMR dataprovided on page 20 herein).

3'-Nitroflavone (VI): ¹ H--NMR (200 Mhz, CDCl₃): δ 8.83 (t, J=1.8 Hz,H-2'), 8.56 (d, J=8.1 Hz, H-4'), 8.42 (dd, J=2.0 Hz, 8.1 Hz, H-5), 8.07(d, J=8.0 Hz, H-6'), 7.88 (m, H-7, H-8), 7.53 (m, H-6), 7.28 (s, H-3).

(2) 6-bromo-3'-nitroflavone (IV)

Steps 1, 2 and 3, same conditions, (Scheme II) but using compounds 6(Aldrich) and (B) as starting materials. ##STR25## [NMR data provided onpage 39 herein](3) 6-bromo-2'-nitroflavone (III)

Steps 1, 2 and 3, same conditions (Scheme II) but using compounds 6 and7 (Aldrich) as starting materials. ##STR26## [NMR data provided on page40 herein](4) 6-bromo-4'-nitroflavone (V)

Steps 1, 2 and 3, same conditions (Scheme II) but using compounds 6 and8 (Aldrich) as starting materials. ##STR27## [NMR data provided on page40 herein](5) 4'-nitroflavone (VII)

Steps 1, 2 and 3, same conditions (Scheme II) but using compounds 2 and8 (Aldrich) as starting materials.

4'-Nitroflavone (VII): ¹ H--NMR (300 Mhz, CDCl₃), δ 8.40 (d, J=8.80 Hz,H-2', H-6'), 8.25 (dd, J=1.60 Hz, 8.0 Hz, H-5), 8.12 (d, J=8.80 Hz,H-3', H-5') 7.77 (td, J=2.0 Hz, 7.20 Hz, H-7), 7.62 (dd, J=1.60 Hz, 8.60Hz, H-8), 7.47 (td, J=1.0 Hz, 8.0 Hz, H-6), 6.92 (s, H-3).

(6) 6-Chloro-3'-nitroflavone (VIII)

Steps 1, 2 and 3, same conditions (Scheme II) but using compounds 1 and9 (Aldrich) as starting materials. ##STR28##

6-chloro-3'-nitroflavone (VIII): ¹ H--NMR (200 Mhz, CDCl₃) δ 8.80 (s,H-2'), 8.41 (d, J=2.5 Hz, H-5), 8.21 (m, H-4', H-6'), 7.75 (t, J=8.2 Mz,H-5'), 7.70 (dd, J=8.7 Hz, 2.4 Hz, H-7), 7.60 (d, J=9.0 Hz, H-8), 6.90(s, H-3).

(7) 6-Fluoro-3'-nitroflavone (IX)

Steps 1, 2 and 3, same conditions (Scheme II) but using compounds 1 and10 (Aldrich) as starting materials. ##STR29##

6-Fluoro-3'-nitroflavone (IX): ¹ H--NMR (200 Mhz, CDl₃) δ 8.82 (t, J=2Hz, H-2'), 8.44 (dt, J=2.1 Hz, 8.2 Hz, H-4'), 8.25 (dt, J=2.2 Hz, 8.3Hz, H-3'), 7.90 (dd, J=3.2 Hz, 8.3 Hz, H-8), 7.80 (t, J=8.1 Hz, H-5'),7.65 (dd, J=3.3 Hz, 8.2 Hz, H-5), 7.52 (m, H-7), 6.91 (s, H-3).

(i) Binding to Central Benzodiazepine Receptors (BDZ--Rs) of RatMembranes (Compound IV).

The binding of ³ H--FNZ (81.8 Ci/mmol; NEN) (FIG. 11) was carried out asdescribed by Levi de Stein, M. et al., Mol. Brain Res. 1989, 5, 9. Inbrief, for each assay, triplicate samples of the membranes, containing0.2 to 0.4 mg protein were suspended in a final volume of 1 mL of 0.25mM Tris--HCl buffer, pH 7.3. The incubation was carried out at 4° C. for60 min with 0.6 nM ³ H--FNZ. to study the binding saturation, a range of0.3 to 10 nM ³ H--FNZ was used. Non-specific binding was determined inparallel incubations in the presence of 3 μM FNZ, and represented 5-15%of total. The assays were terminated by filtration under vacuum throughWhatman GF/A glass-fiber filters, and three washes with 3 mL each ofincubation medium. Filters were dried and counted after the addition of5 mL of 2,5-diphenyloxazole/xylene as scintillation fluid.

(ii) Binding to BDZ--R's of Rat Cerebral Cortex Membranes (Compounds II,III, V, VI, VII, VIII, and IX).

The binding of ³ H--FNZ was carried out as for (i) above.

Results are shown in Table 5 below.

                  TABLE 5                                                         ______________________________________                                        Effect of Compounds II, III, V, VI, VII, VIII and IX                          on .sup.3 H-FNZ Binding to Synaptosomal Membranes from Rat                    Cerebral Cortex.                                                                             Ki                                                                    Compound                                                                              nM                                                             ______________________________________                                               II       20                                                                   III     208                                                                   V       220                                                                   VI      250                                                                   VII     300                                                                   VIII     8                                                                    IX      170                                                            ______________________________________                                    

(iii) Elevated Plus-Maze

The animals used in the pharmacological test were male Swiss mice fromour breeding stock, weighting 28-35 g. They were placed in groups of tenwith free access to water and food, and maintained on 12 h/12 hday/night cycle. The elevated plus-maze set-up consisted of a maze oftwo open arms, 25×5 cm, crossed by two closed arms of the samedimensions, with free access to all arms from the crossing point. Theclosed arms had walls 35 cm high all around. The maze was suspended 50cm from the room floor. Mice were placed on the central part of thecross facing an open arm. The number of entries and the time spend goinginto open and closed arms were counted during 5 min. A selectiveincrease in the parameters corresponding to open arms revels ananxiolytic effect. The total exploratory activity (number of entries inboth arms) was also determined according to the method of Pellow, S. J.et al. Neurosci. Meth. 1986, 14, 149; Lister, R. G. Psychopharmacology1987, 92, 180).

RESULTS AND DISCUSSION

Non-specific nitration of compound 6-bromoflavone yielded three majorproducts. Compounds (III) and (IV) inhibited the binding of ³ H--FNZ toextensively washed rat cerebral cortical membranes with K_(i) of 208±19nM (n=4) and 220±1 nM (n=2) (Table 5), respectively, but compound (IV)was several times more active, as shown in Table 6. The potency ofcompound (IV) in displacing ³ H--FNZ binding was highest in thecerebellum and in one population of cerebral cortical BDZ binding sites(FIG. 11). In the brain regions were the type II BDZ--R predominates,compound (IV) exhibits a potency for displacement of ³ --FNZ binding 3-4times lower than that found for the cerebellum, a region of the brainthat has an homogeneous population of type I BDZ--Rs (Siegharth, W.Trends Pharmacol. Sci. (1992), 13 P. 446; Doble, A. and Martin I. L.Trends Pharmacol. Sci. (1992) 13, p. 76). In addition, in the spinalcord compound (IV) has an affinity 10 times lower than the highest valuefound in the cerebral cortex, where it recognises two distinctpopulations of binding sites with K₁₅ of 1.2 nM and 15.5 nM,respectively (Table 6 and FIG. 11).

                  TABLE 6                                                         ______________________________________                                        Effect of compound IV on .sup.3 H-FNZ binding to synaptosmal                  membranes from several brain structures (see FIG. 11).                        STRUCTURE      n     K.sub.i SEM (nM)                                         ______________________________________                                        cerebellum     4     3.6 ± 0.1                                             hippocampus    2     9.6 ± 0.6                                             striatum       3     9.8 ± 1.6                                             spinal cord    3     12.7 ± 0.5                                            cerebral cortex                                                                              6     1.2 ± 0.4 & 15.5 ± 0.9                             ______________________________________                                    

Scatchard analysis of saturation curves of ³ H-FNZ binding to cerebralcortical membranes reveals that compound (IV) is a competitive ligandfor the BDZ-R (data not shown).

On the other hand, in the cerebellum and the cerebral cortex, compound(IV) displaces ³ H-zolpidem with similar potencies (3±1 nM and 3.8±1 nM,n=3, respectively). Zolpidem is an imidazopyridine possessingselectivity for the type I BDZ-Rs (Arbilla S. et al supra).

Compound (IV) appears to be a selective BDZ-R ligand because it does notdisplace (at 10 μM) ³ H-muscimol, ³ H-AMPA, ³ H-QNB, and ³ H-8-OH-DPATbindings for GABA_(A), AMPA-glutamate, cholinergic-muscarinic andserotonin 1_(A) receptors, respectively.

Preliminary pharmacological experiments in mice reveal that compound(IV) at 0.1 mg/kg, i.p. has anxiolytic properties in the elevatedplus-maze, increasing both the percentage of entries in the open armsand the time spent in these arms (FIG. 12).

In conclusion, we have presented evidence that compound (IV) is a highaffinity BDZ-R ligand with agonistic properties, which recognises twopopulations of binding sites in the cerebral cortex and displays adifferential potency for the inhibition of ³ H-FNZ binding in severalregions of the rat brain, in accordance with the regional distributionof type I BDZ-R.

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What is claimed is:
 1. A method of treating anxiety in a patient whichcomprises administering to the patient an effective non-toxic amount ofa flavonoid of general formula (I): ##STR30## wherein R¹, R², R³, R⁴ andR⁵ are independently selected from NO₂ and H;R⁶ and R⁷ are independentlyselected from Br, Cl, F, and H or R⁶ and R⁷ together form a single bond;R⁸, R¹⁰ and R¹¹ are independently selected from H, --OH, --R, --NO₂, Br,Cl, F, --OR, --NH₂, --NHR, --NR₂, --COOR, --COOH, --CN or a sugar group;R⁹ is selected from H, NO₂, Br, Cl, or F; R is C₁ -C₆ alkyl or alkenyl;with the proviso that at least one of R¹, R², R³, R⁴ and R⁵ is NO₂ andwith the exception that when R⁹ is H, R⁵ is H,or the administration ofan effective non-toxic amount of a bi-flavonoid which is a dimer of acompound of general formula (I).
 2. A method according to claim 1whereinR¹, R² and R⁵ are independently selected from NO₃ and H; R⁸, R¹⁰and R¹¹ are all hydrogen; R⁹ is selected from H, NO₂, Br, Cl, or F; withthe proviso that at least one of R¹, R² and R⁵ is NO₂, with theexception that when R⁹ is H, R⁵ is H.
 3. A method according to claim 1whereinR¹, R² and R⁵ are independently selected from H and NO₂ ; R⁶ andR⁷ together form a single bond; R⁹ is selected from H, NO₂, Br, Cl, orF; with the proviso that at least one of R¹, R² and R⁵ is NO₂ with theexception that when R⁹ is H, R⁵ is H.
 4. A method according to claim 1whereinR¹, R² and R⁵ are independently selected from NO₂ and H; R³, R⁴,R⁸, R¹⁰ and R¹¹ are all H; R⁶ and R⁷ together form a single bond; R⁹ isselected from Br, Cl, F and NO₂ ; with the proviso that at least one ofR¹, R² and R⁵ is NO₂.
 5. A method according to claim 1 wherein thecompound is: ##STR31##
 6. A method according to claim 1 wherein thecompound is:
 7. A method according to claim 1 wherein the compound is:8. A method according to claim 1 wherein the compound is:
 9. A methodaccording to claim 1 wherein the compound is:
 10. A method according toclaim 1 wherein the compound is:
 11. A method according to claim 1wherein the compound is:
 12. A method according to claim 1 wherein thecompound is:
 13. A method according to claim 1 wherein the bi-flavonoiddimer has the general formula (X): wherein R¹ to R¹¹ have the samedefinitions given in claim
 1. 14. A method according to claim 13whereinR¹, R² and R⁵ are independently selected from NO₂ and H; R⁹ isselected from H, NO₂, Br, Cl and F; with the proviso that at least oneof R¹, R² and R⁵ is NO₂ and with the exception that when R⁹ is H, R⁵ isH.
 15. A method according to claim 13 whereinR¹, R² and R⁵ areindependently selected from H and NO₂ ; R⁶ and R⁷ together form a singlebond; R⁹ is selected from H, NO₂, Br, Cl and F; with the proviso that atleast one of R¹, R² and R⁵ is NO₂ and with the exception that when R⁹ isH, R⁵ is H.
 16. A method according to claim 13 whereinR¹, R² and R⁵ areindependently selected from NO₂ and H; R³, R⁴, R⁸, R¹⁰, and R¹¹ are allH; R⁶ and R⁷ together form a single bond; R⁹ is selected from Br, Cl andF and NO₂ ; with the proviso that at least one of R¹, R² and R⁵ is NO₂.17. A method according to claim 1 or 13 wherein the treatment reducesanxiety without exerting a substantially sedative effect.
 18. Apharmaceutical formulation which comprises a flavonoid of formula (I) ora dimer thereof in admixture with a pharmaceutically acceptable carrier##STR32## wherein R¹, R², R³, R⁴ are independently selected from NO₂ andH;R⁸, R¹⁰ and R¹¹ are independently selected from H, --OH, --R, --NO₂,Br, Cl, F, --OR, --NH₂, --NHR, --NR₂, --COOR, --COOH, --CN or a sugargroup; R⁹ is selected from H, NO₂, Br, Cl, or F; R is C₁ -C₆ alkyl oralkenyl; with the proviso that at least one of R¹, R², R³, R⁴ and R⁵ isNO₂ and with the exception that when R⁹ is H, R⁵ is H; or theadministration of an effective non-toxic amount of bi-flavonoid which isa dimer of a compound of general formula (I).
 19. A formulationaccording to claim 18 whereinR¹, R² and R⁵ are independently selectedfrom NO₂ and H; R⁹ is selected from H, NO₂, Br, Cl and F; with theproviso that at least one of R¹, R² and R⁵ is NO₂ and with the exceptionthat when R⁹ is H, R⁵ is H.
 20. A formulation according to claim 18whereinR¹, R² and R⁵ are independently selected from H and NO₂ ; R⁶ andR⁷ together form a single bond; R⁹ is selected from H, NO₂, Br, Cl andF; with the proviso that at least one of R¹, R² and R⁵ is NO₂ and withthe exception that when R⁹ is H, R⁵ is H.
 21. A formulation according toclaim 1 whereinR¹, R² and R⁵ are independently selected from NO₂ and H;R³, R⁴, R⁸, R¹⁰, and R¹¹ are all H; R⁶ and R⁷ together form a singlebond; R⁹ is selected from Br, Cl and F and NO₂ ; with the proviso thatat least one of R¹, R² and R⁵ is NO₂.
 22. A formulation according toclaim 18 wherein the compound of general formula (I) is: ##STR33##
 23. Aformulation according to claim 18 wherein the compound of generalformula (I) is:
 24. A formulation according to claim 18 wherein thecompound of general formula (I) is:
 25. A formulation according to claim18 wherein the compound of general formula (I) is:
 26. A formulationaccording to claim 18 wherein the compound of general formula (I) is:27. A formulation according to claim 18 wherein the compound of generalformula (I) is:
 28. A formulation according to claim 18 wherein thecompound of general formula (I) is:
 29. A formulation according to claim18 wherein the compound of general formula (I) is:
 30. A formulationaccording to claim 18 wherein the bi-flavonoid dimer is of generalformula (X): wherein R¹ to R¹¹ and R have the meaning given in claim 18.31. A formulation according to claim 30 wherein at least one ofR¹ and R²is NO₂ ; R⁹ is selected from H, Cl, Br, F and NO₂ ; R⁶ and R⁷ are both Hor R⁶ and R⁷ together form a single bond; and all other R groups arehydrogen.
 32. A formulation according to claim 30 wherein R¹ and R² areselected from H and NO₂ ;R⁹ is selected from NO₂, Br, F and Cl; and R⁶and R⁷ are both H or R⁶ and R⁷ together form a single bond; and allother R groups are hydrogen.
 33. A formulation according to claim 32wherein the bi-flavonoid dimer is a dimer of monomers selected fromcompounds II, III, IV, V, VII, VIII, or IX.
 34. A flavonoid compound ofgeneral formula (I): ##STR34## wherein R¹, R², R³, R⁴ and R⁵ areindependently selected from NO₂ and H;R⁸, R¹⁰, and R¹¹ are independentlyselected from H, --OH, --R, --NO₂, Br, Cl, F, --OR, --NH₂, --NHR, --NR₂,--COOR, --COOH, --CN or a sugar group; R⁹ is selected from H, NO₂, Br,Cl, or F; R is C₁ -C₆ alkyl or alkenyl; with the proviso that at leastone of R¹, R², R³, R⁴ and R⁵ is NO₂ and with the exception that when R⁹is H, R⁵ is H; or a bi-flavonoid which is a dimer of a compound ofgeneral formula (I).
 35. A compound according to claim 34 whereinR¹, R²and R⁵ are independently selected from NO₂ and H; R⁸, R¹⁰ and R¹¹ areall hydrogen; R⁹ is selected from H, NO₂, Br, Cl and F; with the provisothat at least one of R¹, R² and R⁵ is NO₂ and with the exception thatwhen R⁹ is H, R⁵ is H.
 36. A compound according to claim 4 whereinR¹, R²and R⁵ are independently selected from H and NO₂ ; R⁶ and R⁷ togetherform a single bond; R⁹ is selected from H, NO₂, Br, Cl and F; with theproviso that at least one of R¹, R² and R⁵ is NO₂ and with the exceptionthat when R⁹ is H, R⁵ is H.
 37. A compound according to claim 34wherein:R¹, R² and R⁵ are independently selected from NO₂ and H; R³, R⁴,R⁸, R¹⁰, and R¹¹ are all H; R⁶ and R⁷ together form a single bond; R⁹ isselected from Br, Cl and F and NO₂ ; with the proviso that at least oneof R¹, R² and R⁵ is NO₂.
 38. A flavonoid compound according to claim 36which is selected from compounds II, III, IV, V, VI, VII, VIII and IX.39. A flavonoid compound according to claim 38 which is selected fromcompounds II, IV, VIII.
 40. The compound: ##STR35##
 41. The compound:42. The compound: